1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing L-aspartic acid or L-aspartic acid-derived metabolites using a bacterium of Enterobacteriaceae family which has been modified to have aspartate dehydrogenase.
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to feedback inhibition by the resulting L-amino acid (U.S. Pat. Nos. 4,346,170, 5,661,012 and 6,040,160).
L-aspartic acid is a useful metabolite having different applications (aspartame sweetener, biodegradable polymers, etc.). In the currently used industrial process, aspartic acid is produced enzymatically from fumarate using aspartase. In turn, fumarate can be obtained by chemical synthesis, as a product of oil distillation or from hydrocarbons in bacterial fermentation process.
A known three-step production process includes the steps of: 1) production of calcium-fumarate in fermentation of a bacterial strain; 2) conversion of the calcium-fumarate to diammonium fumarate by addition of ammonia, ammonium carbonate or ammonia in combination with CO2; and 3) enzymatic conversion of the obtained diammonium fumarate to ammonium aspartate by aspartase (U.S. Pat. No. 6,071,728).
Direct methods for producing L-aspartic acid from carbohydrates or hydrocarbons in bacterial fermentation have been previously developed using bacteria belonging to the genus Corynebacterium or Brevibacterium (U.S. Pat. No. 4,000,040). Some wild-type strains of these genera are the natural producers of L-aspartic acid. These methods and bacteria relate to the process of selecting the induced mutants with increased producing ability.
It has been shown by Yang Zh. et al (J. Biol. Chem., 278(10): 8804-8808 (2003)), that aspartate dehydrogenase isolated from Thermotaoga maritima can catalyze the forward reaction of aspartate synthesis from oxaloacetate and ammonia in vitro. Use of this reaction for aspartate synthesis could help to avoid the glutamate by-production. On the other hand, the authors have proposed that the role of this enzyme in vivo is the conversion of aspartic acid to iminoaspartic acid, the precursor of NAD biosynthesis. Moreover, it has been shown that the aspartate dehydrogenase from T. maritima has a high optimum temperature (+70° C.) and saves only low residual activity at +30° C.-+40° C.
But currently, there have been no reports of using a bacterium of Enterobacteriaceae family modified to have aspartate dehydrogenase for producing L-aspartic acid or L-aspartic acid-derived metabolites.